Self-renewal and differentiation of neural stem (progenitor) cells (NSCs) is accurately controlled during embryogenesis by secreted molecules such as Wnt proteins. Misregulated Wnt signaling is implicated in various neurological diseases. However, the mechanisms underlying Wnt signaling in the nervous system are unknown. We identified the Ryk protein as a non-Frizzled Wnt receptor and demonstrated that its activity is required for corticogenesis. The Ryk protein is cleaved by 3- secretase (presenilin) and the released Ryk intracellular domain (ICD) translocates to the nucleus following Wnt3 stimulation, where it interacts with a novel nuclear protein, Arip. Arip over- expression increases Ryk ICD nuclear localization and neurogenesis. Preliminary data shows that Ryk ICD and Arip form a complex with transcriptional coactivators and corepressors. We hypothesize that Ryk signaling functions through Arip to regulate neurogenesis. This central hypothesis underlies the following specific aims. Aim 1 will determine the role of Arip in neurogenesis in vivo. We generated Arip1 and Arip2 knockout mice and will determine their neurogenesis phenotypes. We will also assess potential genetic interaction of Ryk and Arip using Ryk and Arip knockout mice. In addition, we will perform in utero electroporation experiments to determine whether Ryk ICD/Arip synergy promotes neurogenesis as well as the epistatic relationship between Ryk and Arip. In Aim 2, we will investigate how Wnt signaling and Arip regulate Ryk ICD nuclear signaling. We hypothesize that Arip is required for Ryk ICD nuclear localization regulated by Wnt signaling. We will determine if Wnt-induced Ryk ICD nuclear localization requires Arip, and if Wnt signaling regulates Arip transcription or protein stability. Aim 3 will define the molecular mechanisms underlying Arip and Ryk ICD transcriptional activity. We will determine if Arip is required for Ryk ICD-mediated regulation of target genes. In addition, we will determine if Ryk ICD and Arip are recruited to promoters of downstream target genes. This study should provide new insights into the molecular mechanisms governing differentiation of neural stem cells. Understanding the basic mechanisms of cell signaling in neural stem cells may help us develop more effective therapeutic treatments for developmental neurological diseases. The knowledge gained from the proposed studies will also be useful for stem cell application in therapy to treat degenerative neurological diseases. PUBLIC HEALTH RELEVANCE: This study should provide new insights into the molecular mechanisms governing self- renewal and differentiation of neural stem cells. As many developmental neurological diseases result from defects in this process, understanding the basic mechanisms of self-renewal and differentiation in neural stem cells may help us develop more effective therapeutic treatments. The knowledge gained from the proposed studies will also be useful for cell replacement therapy to treat degenerative neurological diseases, such as Alzheimer's disease, Parkinson's disease and Huntington's disease.